The traditional view of mitochondria as isolated, spherical, energy producing organelles is undergoing a revolutionary transformation. Emerging data show that mitochondria form a dynamic networked reticulum that is regulated by cycles of fission and fusion. The discovery of a number of proteins that regulate these activities has led to important advances in understanding human disease. We have demonstrated that activation of dynamin related protein 1 (Drp1), a protein that controls mitochondrial fission, is reduced following exercise in prediabetes, and the decrease is linked to increased insulin sensitivity and fat oxidation. We now propose to build on this research and test the hypothesis that mitochondrial dynamics is a key mechanism of insulin resistance in type 2 diabetes. Our central hypothesis is that in diabetes elevated mitochondrial lipid metabolism causes recruitment and activation of Drp1 - likely through increased reactive oxygen species, leading to increased mitochondrial fragmentation and opening of the mitochondrial permeability transition pore. In Aim 1a we will perform in vivo and in vitro studies of human skeletal muscle mitochondrial dynamics across the metabolic phenotype ranging from patients with type 2 diabetes, to obese, to lean healthy controls. Translational first-in-man studies will use an acute lipid challenge (Aim 1b) and exercise training (Aim 1c) to investigate the physiological significance of altered skeleta muscle mitochondrial dynamics on insulin sensitivity in humans. Insulin resistance will be assessed using euglycemic hyperinsulinemic clamps, and in vivo substrate metabolism will be measured using indirect calorimetry. Mitochondrial fission/fusion, fragmentation, function, membrane potential, mitochondrial reactive oxygen species, and the accumulation of lipid intermediates will be assessed from muscle biopsy tissue and permeabilized muscle fibers. In Aim 2, we will use inhibition and expression cloning experiments to directly examine the impact of manipulating mitochondrial fragmentation in intact ex vivo cultured human skeletal muscle cells. This research will provide a comprehensive and complementary analysis of skeletal muscle mitochondrial dynamics, and will also generate novel data on the link between exercise and nutrient regulation of mitochondrial dynamics and function in type 2 diabetes. The experimental approach harnesses innovative molecular and cellular tools, interfaced with physiologically significant human studies to obtain meaningful data on insulin resistance, and has the potential to generate insights that will lead to new diabetes therapies for future generations.